Project Summary The long-term goal of this project is to define the cellular and molecular mechanisms underlying hemodynamic abnormalities in glaucoma and to find a means to mitigate retinal ganglion cell damage. In this project, we will test a novel hypothesis that astrocytes are central to pressure-initiated ocular BF autoregulation, as they sense transmural pressure and tissue oxygen levels to coordinate the perivascular astrocytic network and feedback to vessels to modulate diameter and thus blood flow and that failure of this mechanism results in hemodynamic imbalance. The hypothesis will be tested in rat retina and monkey optic nerve head in three specific aims: (1) Pressure-initiated retinal astrocyte activation is independent of neuronal and metabolic activity. This will be achieved by elimination of retinal ganglion cell and application of systemic hyperoxia. (2) Astrocytes modulate retinal vascular tone and coordinate pressure-initiated autoregulation under modulation by tissue oxygen status. To test the hypothesis, all or a specific pathway of the proposed feedback mechanism in the astrocytes will be inhibited and tested under hyperoxia and normoxia to compare the hemodynamic parameter changes under the conditions. (3) Pharmacological interruption of astrocyte feedback mechanism impairs hemodynamic balance within the optic nerve head and this is further modified by the alteration of oxygen supply. The following techniques are used: (1) an ex vivo system that enables precise control of intravascular and extravascular pressures thus allowing simultaneous quantifying astrocytic Ca2+ (Fluo-4AM) and changes in vascular diameter. (2) In vivo imaging of astrocytic Ca2+ (Fluo-4AM, fluorescence) and vessel diameter (infrared) using a confocal scanning laser ophthalmoscope, while blood pressure and intraocular pressure are manipulated in both rats and nonhuman primates. (3) Combining pharmacological inhibitors and adeno-associated virus 8 vector delivery with an astrocyte-specific promotor inhibit either all the bioactivity of astrocytes or the key proteins in the proposed astrocytic feedback and propagation pathways. (4) In vivo assessment of BF autoregulation in retina and optic nerve by dynamic autoregulation analysis using laser speckle imaging techniques. The proposal is expected to elucidate a novel role for astrocytes in pressure-initiated BF autoregulation, thus significantly expanding our understanding of hemodynamic control in the central nervous system. This basic understanding provides a platform for future studies of novel therapeutic targets in diseases associated with autoregulation dysfunction, including glaucoma.